Ribonuclease P (RNase P) is a ribonucleoprotein complex responsible for processing many different RNA molecules in the cell. It is found in almost all organisms, from bacteria to humans, and is composed of one essential RNA subunit and one or more protein subunits. The RNA component is responsible for catalysis as it can process RNA in vitro in the absence of protein. The only common RNase P function in all organisms is the 5' end maturation of transfer RNA (tRNA). RNase P was one of the first catalytic RNA molecules discovered and its study has been pivotal to our understanding of the role of RNA molecules in catalysis. RNase P is a true multi-turnover ribozyme that recognizes its substrate in trans and one of only two universal ribozymes. The knowledge of the structure and function of RNase P promises to provide important and relevant information on a key ribozyme involved in a central cellular process common to all organisms and also to further our understanding of the structure and function of large RNA molecules. This proposal is concerned with the structure and function of RNase P. In the past few years we have made substantial progress in our studies, including solving the structure of the complex formed by Thermotoga maritima RNase P holoenzyme and mature tRNA. The structural studies of the holoenzyme/tRNA complex show that all RNase P ribozymes share a common, RNA-based mechanism of RNA cleavage and recognition where the protein component increases RNase P functionality by accurately positioning the 5' leader pre-tRNA substrate and by contacting conserved regions of the P RNA structure. The structure also shows that RNase P utilizes shape complementarity, specific RNA-RNA contacts, and intermolecular base pairing to recognize its substrate efficiently and that both P RNA and the pre-tRNA help to coordinate two catalytically important metal ions essential for the mechanism of pre-tRNA cleavage. For the next period we propose to continue and expand our structural studies of RNase P. The specific aims for this proposal are: 1) to determine the three dimensional structure of complexes of RNase P holoenzyme with different substrates, 2) to determine the structure of complexes of RNase P holoenzyme with pre-tRNA and transition state analogues and, 3) to study the role of the universally conserved regions in the structure of RNase P and also the role of important amino acids involved in leader recognition. The work is based on a combination of molecular biology and biochemical methods to produce and characterize the molecules that we require for our work and X-ray crystallography to solve their atomic structures. The work on RNase P has important implications for health related studies. RNase P is a promising target for the development of new chemotherapeutics as the specificity of RNase P can be altered to create molecules that degrade target RNA molecules. PUBLIC HEALTH RELEVANCE: We propose to study the structure and mechanism of bacterial ribonuclease P (RNase P), a universal ribozyme that is responsible for tRNA maturation and processing of other RNA molecules. The long term goal of our work is to provide a comprehensive understanding of the way this crucial ribonucleoprotein complex recognizes and processes RNA, the details of the chemical mechanism of action, and the role of different regions in catalysis and RNA recognition.